Control of Hedgehog Signal Transduction by Neuropilin

描述：Hedgehog(HH)信号被用于控制大多数发育中的组织和器官中的细胞命运，以及在许多再生事件中。HH信号的缺陷会导致出生缺陷和癌症。关于HH信号是如何传递的，许多机制上的谜团仍然存在。通过高通量RNAi筛选，我们确定Neuropilins(NRP)1和2是脊椎动物HH信号的新的、特异的调节因子。NRPs是一种单程跨膜蛋白，参与多种分泌配体的接收，包括信号素和VEGF165，并参与细胞黏附和细胞迁移。在成纤维细胞中，阻断NRPs对HH信号转导的抑制作用与阻断纤毛形成或阻断平滑功能的作用相当。相反，任一种NRP的过量产生都会使细胞对HH信号敏感。HH途径的新成分很少被发现；NRP可能由于其部分冗余而被遗漏。我们发现了两种蛋白质，它们的功能是这一重要的形态发生途径所必需的，这可能会从根本上改变目前的HH信号转导模型，并扩大对NRP在其他信号转导途径中功能的理解。目的1.确定NRPs调节HH信号转导的机制。NRP可以通过直接与已知的HH途径成分结合，或介导与HH信号转导融合的其他信号，或通过改变HH信号转导所需的细胞属性或过程来影响HH信号转导。我们将通过确定HH信号中的哪些步骤受到影响，NRPs对HH信号的影响是否涉及细胞黏附或迁移变化，以及哪些蛋白质直接与NRPs相互作用来研究这些可能性。目的2.确定NRP的哪些结构域需要支持HH信号，以及已知的NRP共受体、配体或效应分子是否能够交叉调节HH通路。我们将通过两种方式研究哪些结构域对HH信号转导起作用：工程化的结构域缺失，以及高通量筛选干扰NRP对HH信号转导的支持的点突变。NRP转导血管内皮生长因子和信号素信号，分别作为血管内皮生长因子受体和神经丛蛋白的共同受体。我们将测试血管内皮生长因子、血管内皮生长因子受体、丛状蛋白受体或信号素是否参与NRPs对HH信号转导的影响。目的3.研究NRPs如何影响HH依赖的发育和肿瘤的发生。利用携带两个NRP基因突变的小鼠，我们将控制时间和组织特异性的NRP功能的移除，以测试它们在体内几个依赖HH的发育过程中的参与。使用新创造的慢病毒，它编码阻止NRP基因的特定抑制RNA，我们将感染HH反应细胞的原代培养，并监测对HH靶基因表达的影响。

DESCRIPTION (provided by applicant): Hedgehog (Hh) signaling is employed in controlling cell fates in most developing tissues and organs, as well as during many regeneration events. Defects in Hh signaling lead to birth defects and cancer. Many mechanistic mysteries remain regarding how an Hh signal is transduced. Using high-throughput RNAi screening, we identified Neuropilins (Nrp) 1 and 2 as novel, specific regulators of vertebrate Hh signaling. Nrps are single-pass transmembrane proteins implicated in the reception of a diverse set of secreted ligands, including Semaphorins and VEGF165 and in cell adhesion and cell migration. In fibroblasts the inhibition of Hh signal transduction resulting from blocking Nrps is as strong as the effect of blocking cilia formation or blocking Smoothened function. Conversely, over-production of either Nrp sensitizes cells to Hh signals. New components of the Hh pathway are uncovered infrequently; the Nrps were probably missed due to their partial redundancy. Our discovery of two proteins whose functions are required by this important morphogenic pathway has the potential to bring fundamental changes to current models of Hh signaling and to enlarge the understanding of Nrp functions in other signaling pathways. Aim 1. Determine the mechanism by which Nrps regulate Hh signal transduction. Nrps could influence Hh signal transduction by directly associating with known Hh pathway components, or mediating other signals that converge with Hh transduction, or by altering cell properties or processes that are required for Hh transduction. We will investigate each of these possibilities by determining which steps in Hh signaling are affected, whether cell adhesion or migration changes are involved in the effect of Nrps upon Hh signaling, and what proteins interact directly with Nrps. Aim 2. Determine which domains of Nrp are needed to support Hh signaling, and whether known Nrp co-receptors, ligands, or effector molecules are capable of Hh pathway cross- regulation. We will investigate which domains contribute to Hh signal transduction in two ways: engineered domain deletions, and a high-throughput screen for point mutations that interfere with Nrp support of Hh signal transduction. Nrps transduce VEGF and Semaphorin signals, acting as co-receptors for VEGF receptors and Plexins, respectively. We will test whether VEGF, VEGF receptor, Plexin receptors, or Semaphorins are involved in the effect of Nrps upon Hh signal transduction. Aim 3. Investigate how Nrps influence Hh- dependent development and tumorigenesis. Using mice that carry mutations in both Nrp genes, we will control temporal and tissue-specific removal of Nrp functions to test their involvement in several Hh-dependent developmental processes in vivo. Using newly created lentiviruses, which encode specific inhibiting RNAs that block the nrp genes, we will infect primary cultures of Hh-responsive cells and monitor effects on Hh target gene expression.